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  • C12N15/09—Recombinant DNA-technology
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  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
• • C—CHEMISTRY; METALLURGY
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

• TMM is an artificial medium for the culturing of tomato embryos the composition of which is illustrated in Table B.
• a "protoplast” is a plant cell from which the cell wall has been removed, usually by enzyme digestion, but which is bounded by a cell membrane;
• “meristem” or “meristematic tissue” is comprised of cells which are fully capable of further division, giving rise in turn to embryonic, primary or secondary tissue;
• “genome” as used herein refers to all of the genetic material found in a cell, both chromosomal and extra-chromosomal;
• a cell “transformed” by DNA is a cell which contains said DNA or a descendant thereof through mitosis or meiosis, which still retains said DNA sequence in its genome.
• “membrane-permeating” agents are agents known as such for mammalian cells;
• the present invention relates to a method for transforming plant cell material comprising contacting the cell material with a transform­ation solution comprising DNA and a cell membrane-permeating agent in the presence of an electrical current for a time sufficient to effect transformation.
• the method of this invention has numerous advantages. It allows the penetration of numerous layers of cells such that whole tissues rather than single cells may be used. The cells are not injured; they maintain their viability and can be used for the production of mature, fertile plants. The method also allows for the transformation of plants which had previously not been successfully transformed.
• the invention also relates to a method for producing a transformed fertile plant comprising contacting plants cells with a transformation solution comprising DNA and a cell membrane-permeating agent in the presence of an electric current for a time sufficient to effect transformation and culturing the thus transformed plant cells under culture conditions.
• the present invention utilises a more constantly, longer applied electromotive force (tension) which effects a carrying or trans­porting of the DNA to and through the cell membrane, the permeability of which is sufficiently enhanced by the membrane-permeating agent.
• any non-protoplastic plant material may be utilised.
• the present inven­tion envisages the transformation of plant tissue, plant embryo, meri­stematic tissue such as tassel or ear meristem, axillary buds, stem strips, callus or cell suspensions.
• plant embryonic tissue it is preferred that the embryos have reached stage 3 of development as defined by Abbe and Stein in Am J Botany 41 (1954) 286-287, ie at a stage between 22 and 28 days after pollination.
• a corn embryo at stage 3 is typically about 3mm long and he scutellar node tissue is extern­ally recognisable by the two constrictions which set it off.
• the primary root has become recognisable and within the coleoptile the first and second leaves have increased considerably in size. This stage is characterised by the third leaf primordium which has recently arisen.
• the plant material employed is conveniently in excised form.
• the process of the invention is conveniently carried out in a hori­zontal gel electrophoresis system, eg an agarose gel electrophoresis system, into which the plant material to be transformed and the DNA are placed in wells such that the electric current will run from the DNA to the plant material.
• a hori­zontal gel electrophoresis system eg an agarose gel electrophoresis system
• the embryo is positioned in the well such that the side containing the meristematic tissue is facing the wells containing the transformation solution.
• a solution containing the DNA (which may, to facilitate selection at a later stage, contain, for example, a DNA sequence coding for resistance to a specific antibiotic) and a membrane-permeating agent are then placed in wells adjacent to those containing the plant cell material.
• Preferred membrane-permeating agents are polar, such that they are car­ried, in association with the DNA, to and through the cell membrane when the tension is applied.
• membrane-permeating agents include DMSO, lysolecithin and detergents such as sodium dodecyl sul­phate and Triton-X, preferably DMSO.
• concentrations of such a mem­brane-permeating agent uses should be sufficient to render the cell membrane temporarily permeable but without destroying the integrity of the intracellular organelle membranes. Such concentrations may vary within wide ranges and will i.a. depend on the plant material involved. Tougher plant cell material, such as dicotyledonous cell material may stand, for example, a concentration of 10% DMSO.
• a concentration of membrane-permeating agent of from about 1% to about 4%, preferably about 2%, by weight of the transformation solution.
• a tracking dye may optionally be included in the transformation solution to enable the progress of the vectors to be monitored. Examples of suitable tracking dyes would be bromophenol blue, bromocresol green or xylene cyanol. Such dyes and their use for such a purpose are well known in the art.
• a low voltage electric curr­ent is applied such that it runs in a direction from the vector-contain­ing wells toward the wells containing the plant material.
• the tissues are washed with sterile liquid and placed on solid medium in order for them to recover from any trauma associated with the electro­transformation process.
• the period of time spent on the solid medium is dependent on the age and size of the tissues treated and younger and/or smaller tissues may require a longer recovery period prior to the selec­tion process.
• the tissues are normally kept on the medium for 2-3 days, after which the successfully transformed tissues are selected, which process may, if the transformation vector containing a DNA sequence co­ding for resistance to a specific antibiotic, be by exposure of the tissues to this specific antibiotic. Those tissues which germinate (develop roots and shoots in the case of embryos) or grow and develop chlorophyll on the selection medium have acquired resistance to the antibiotic by uptake and incorporation of the transforming DNA.
• the voltage (tension) applied should be sufficient to assist in the transportation of the DNA and not exceed a tension which would substan­tially damage the cells.
• the tension to be employed may vary within wide ranges and will depend on various factors such as the type and form of the plant material employed and the time during which the tension is applied.
• 50% of corn embyros will survive when put under a tension of 200V for 5 minutes in a gel electrophoresis system as disclosed in Example 1. (Corn is about the toughest monocot, and dicots survive, in general, better than monocots.) Voltages up to 110V may be used without seriously damaging the embyro, although germination may be delayed. Voltages above 200V should in general be avoided.
• transformation may still be obtained with a tension as low as 8V.
• an electric tension from 40V to 140V, eg from 50V to 140V, preferably from about 50V to 110V and most preferably from 52V to 80V.
• the electrodes are 18cm apart.
• the application of a tension of eg 50V in such a system will result in an electric field strength of 50V/18cm or 2.78V/cm.
• the electric current strength produced in such systems will depend i.a. on the trans­formation solution (gel etc) and the voltages applied.
• the electro­phoresis system employed in Example 1 at the voltage of 52V an electric current of circa 25mA is produced.
• the period of time of exposure to the electric current is dependent on various factors such as the distance between the wells containing the tissue and the wells containing the transforming solution and also on the size of the tissue sample.
• the minimum period of time is that necessary for the DNA in the transforming solution to flow, under the influence of the electric field, into the area of the gel containing the plant material samples.
• the distance between the two sets of wells is 1 mm at initiation of the current, at a voltage of 52V it will take about 13 to 15 minutes for the transforming solution to move past a corn embryo and it will take about 20 minutes for the solu­tion to move past a larger tissue such as a tassel initial.
• transformation will be obtained in good yields after exposure to the electric tension for 5 to 25 minutes, in particular for 10 to 20 minutes.
• the distance between the set of wells ie those containing the tissue sample and those containing the transforming solution, is depen­dent on the strength of the transforming solution and the concentration of the support gel such that the distance is preferably higher when the gel is more dilute.
• the DNA to be employed is conveniently in vector form, preferably in plasmid form, which plasmid is genetically manipulated using standard recombinant DNA techniques well known to those skilled in the art, so as to contain DNA with which it is desired to transform the plant tissue.
• a preferred class of DNA for use in this invention can be classified as foreign DNA.
• foreign DNA refers to any DNA originating from a source other than the host or recipient species.
• Foreign DNA includes for example, non-host plant DNA, synthetic DNA sequences, sequences produced by recombinant DNA techniques, bacterial, fungal, viral, animal DNA sequences and the like.
• Suitable foreign DNA can include non-host plant promoters such as T-DNA promoters from Ti and Ri plasmids, plant virus promoters such as CaMV, TMV, BMV etc, and the like.
• Valuable foreign DNA can also include foreign structural sequences from the genes for, eg chloramphenicol acetyl transferase (CAT), neomycin phosphotransferase II (npt-II), nop­aline synthase (nos), ⁇ -galactosidase ( ⁇ -gal), the glyphosate resistance gene (EPSP which is the enzyme conferring resistance to glyphosate-5-­enolpyruvylshikimate-3-phosphate synthase) and Bacillus thuringiensis type genes coding for a crystal protein insect toxin.
• CAT chloramphenicol acetyl transferase
• npt-II neomycin phosphotransferase II
• nos nop
• the aformentioned Helio­this , Spodoptera , and Diabrotica species are pests infecting crops such as corn.
• Foreign DNA also includes synthetic genes, such as synthetic DNA sequences based upon native host plant genes, eg a zein gene altered so as to change the amino acid composition of the maize storage protein.
• the foreign DNA of the present invention preferably comprises chimeric constructions such as heterologous promoter/structural sequence combin­ations.
• heterologous constructions include the Bt toxin gene under the control of the ADH promoter, and selectable markers such as the CAT or npt-II structural sequence under the control of a T-DNA promotor or a CaMV promoter.
• selectable markers such as the CAT or npt-II structural sequence under the control of a T-DNA promotor or a CaMV promoter.
• Such combinations may be constructed according to standard recombinant techniques such as those illustrated in Maniatis et al, Molecular Cloning: A Laboratory Manual (1982) and DNA Cloning Vol I & II (D Glober, Ed 1985).
• Other DNA sequences or com­binations of such will be readily apparent to one of ordinary skill in the art.
• the plant material is transformed by a selectable marker gene so that identification of successfully trans­formed tissues is facilitated.
• marker genes are, for example, genes coding for resistance to antibiotics (such as kanamycin, hygromycin, neomycin and chloramphenicol), genes for herbicide resistan­ce and genes for colour (eg the anthocyanin gene).
• antibiotics such as kanamycin, hygromycin, neomycin and chloramphenicol
• genes for herbicide resistan­ce genes for colour (eg the anthocyanin gene).
• the presence of a gene coding for an antibiotic will, for example, enable transformed plant cells to survive and grow in a medium containing the selective antibiotic.
• the plant material is transformed by genes coding for traits which have high commercial or agricultural value, such as resistance to insects, resistance to herbicides, resis­tance to viruses, resistance to fungi, or for enzymes which will inter­fere in particular biochemical pathways, such as those leading to the synthesis of essential amino acids.
• the invention further provides healthy, fertile monocotyledonous plants comprising in their genome DNA originating from a source other than the host or recipient species and parts thereof, in particular seeds of such plants.
• the plasmids H83E and H83R each con­tain the pUC 8 plasmid with a cauliflower mosaic virus (CaMV) 35S promo­ter [nucleotides 7013 to 7436, see Hohn et al, Curr Top Microbiol Immu­nol 96 (1982) 193] a hygromycin phosphotransferase (HPT) coding sequence [the Bam H1 fragment from pLG83, see Gritz and Davies, Gene 25 (1983) 179] and a nopaline synthase (NOS) terminator [nucleotides 682 to 437, see Bevan et al, Nucleic Acids Res 11 (1983) 369].
• the HPT sequence is in the sense orientation relative to the promoter and terminator sequen­ces in plasmid H83E and in the antisense orientation in plasmid H83R.
• Each gel is placed in a horizontal gel electrophoresis system (BRL H6) with 450 ml of sterile tris-acetate-EDTA running buffer (pH 8.0). Gels are exposed to an electric current of 52V, running in the direction from the DNA to the embryos, for a period of either 10, 12.5 or 15 minutes. After exposure, the embryos are rinsed with sterile CM(30) and placed on solid CM(30) medium for 3 days. The embryos are then trans­ferred to CM(30) medium containing 100 ⁇ g/ml of hygromycin. The embryos are scored at 11 and 14 days after electrotransformation treatment, with the following results:
• the green germlings (seedlings) from each test group in Example 1 are collected and ground up, and the nucleic acid is extracted following the cetyltrimethylammon­ium bromide (CTAB) procedures described by Rogers et al Plant Mol Biol 5 : (1985) 69.
• CTAB cetyltrimethylammon­ium bromide
• Leaves from two hygromycin-selected Brassica were cut into pieces and subjected to secondary selection on BSS medium containing 25 ⁇ g/ml hygromycin. These pieces remained green and formed shoots. When they were transferred to BSS with half strength salts and no hygromycin they also rooted. Leaves of the same two primary transformants were also analysed for the presence of the hygromycin gene. Each leaf (approx 0.2g) was frozen in liquid nitrogen and ground to a powder in a mortar. To this, 15ml ice cold sucrose buffer (containing 15% sucrose, 50mM Tris pH 8.0, and 50mM EDTA) and 0.25M NaCl was added to the tissue in the mortar and grinding continued.
• sucrose buffer containing 15% sucrose, 50mM Tris pH 8.0, and 50mM EDTA
• Plasmid pZ033 contains a CaMV 35S promoter (nucleotides 7069 to 7569) in the SacI site of pUC19 (commercially available from Pharmacia and from US Biochemical), the 773 bp chloramphenicol acetyl transferase (CAT) gen from Tu9 [see, Alton and Vapnek, Nature 282 (1979) 864] whose TagI ends are changed to PstI, in the Pst site, and a NOS terminator (nucleotides 682 to 437) between the PstI and the Hind III sites.
• CaMV 35S promoter nucleotides 7069 to 7569
• CAT chloramphenicol acetyl transferase

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